Project Summary Mller glia, which closely resemble retinal progenitor cells (RPCs) in their morphology and gene expression profile, are the last cell type generated in the developing retina. Mammalian Mller glia, however, rapidly lose the ability to proliferate and generate neurons following specification. The molecular mechanisms that allow RPCs to become gliogenic, and lead Mller glia to lose neurogenic competence remain poorly understood. We have recently identified the LIM homeodomain factor Lhx2 as a central regulator of MG differentiation and function, and now seek to characterize its mechanism of action. Based on our preliminary findings, we hypothesize that Lhx2 cooperates with NFI and SoxE factors to initiate gliogenesis. We propose that NFI and SoxE factors bind to target sequences that control expression of gliogenic and glial-specific genes in late-stage RPCs, which then exposes binding site for Lhx2. Lhx2 then induces chromatin opening at these sites, which in turn leads to stable activation of these genes. In postmitotic glial precursors, in contrast, rising levels of Rnf12 inhibit Ldb1/Lhx2-dependent transcriptional activation of neurogenic bHLH genes, eventually leading to a permanent loss of neurogenic competence in mature Mller glia. We aim to determine how NFI, SoxE, and Lhx2 act to drive glial differentiation and restrict neurogenic competence. We will first determine whether NFI factors promote gliogenic competence and glial differentiation in retina. We will next test whether NFI and SoxE directly regulate expression of gliogenic and glial-specific genes, and determine whether selective loss of Lhx2 in differentiating glia reduces chromatin accessibility at cis-regulatory elements that control glial-specific genes. We will also test whether NFI and SoxE direct target site selection by Lhx2 in late- stage RPCs and glial precursors. In addition to these studies of how glial differentiation is controlled, we intend to determine how Lhx2-dependent neurogenic competence is lost in differentiating Mller glia. We will determine whether Rnf12 acts in an Lhx2-dependent manner to drive loss of neurogenic competence in differentiating and mature Mller glia. Finally, we will identify molecules that mediate Lhx2-dependent transcriptional repression in postnatal retina. We anticipate that a full understanding of the mechanism by which Lhx2 regulates gliogenesis will identify key targets for drug and gene-based therapies aimed at restoring vision through controlled dedifferentiation of Mller glia.